Pesticide Biochemistry and Physiology 101 (2011) 175–181

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Pesticide Biochemistry and Physiology

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Protective role of propolis against reproductive toxicity of chlorpyrifos in male rats

Reda H. ElMazoudy a, Azza A. Attia a, Nahla S. El-Shenawy b,c,⇑a Zoology Department, Faculty of Science, Alexandria University, Alexandria, Egyptb Biology Department, Faculty of Science, Taif University, Taif, Saudi Arabiac Zoology Department, Faculty of Science, Suez Canal University, Ismailia, Egypt

a r t i c l e i n f o a b s t r a c t

Article history:Received 15 March 2011Accepted 7 September 2011Available online 1 October 2011

Keywords:ChlorpyrifosPropolisReproductive toxicityTetsosteroneTestesSpermatozoaBiochemical

0048-3575/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.pestbp.2011.09.003

Abbreviations: b.w., body weight; AST, aspartatetransaminase; AlP, alkaline phosphatase; LDH, lactate⇑ Corresponding author at: Zoology Department, F

University, Ismailia, Egypt.E-mail address: elshenawy_nahla@hotmail.com (N

Chlorpyrifos (CPF) is causing different toxicity and propolis has been reported to be an effective antiox-idant. Therefore, the present study is aimed to elucidate the possible protective effects of propolis in alle-viating the toxicity of chlorpyrifos on reproductive performance, testosterone levels, enzyme activitiesand lipids profile in serum of male rats. Animals were divided into four groups; one orally administered(o.d.) CPF at a dose of 9 mg/kg b.w. for consecutive 70-days, propolis group (50 mg/kg b.w.), propolis andchlorpyrifos group and a control group. Results showed that there was a correlation between CPF admin-istration and the significant decrease of the sperm counts, spermatozoon survival and testosterone levelas well as increase of sperm aberrations. CPF increased significantly the lipid profile and the levels of var-ious serum liver marker enzymes. In contrast, co-administration of propolis to CPF-treated rats restoredalmost most of these biochemical parameters to normal levels. On the other hand, CPF resulted in histo-pathological alterations in testes of male rats. However, pre-administration of propolis to CPF-treatedanimals improved the testicular damage and alleviates the toxic effects of CPF on reproductive functionsin male rats.

� 2011 Elsevier Inc. All rights reserved.

1. Introduction

Organophosphates are among the most widely used syntheticinsect pesticides. The widespread use of organophosphates hasstimulated research into the possible existence of effects relatedwith their reproductive toxic activity [1]. Occupational exposuresto pesticides could diminish or destroy the fertility of workerssparked a concern about the effects of hazardous substances onmale reproductive health. The issue of testicular toxicity is ofgrowing concern as a large number of Organophosphates viz., diaz-inon [2], and methyl parathion [3] adversely affect the testicularfunctions in experimental animals. Owing to the extensive use oforganophosphate pesticides in agriculture there is a high risk ofhuman exposure to these chemicals [4].

Chlorpyrifos (CPF), (O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl)phosphoro-thioate) is a conventional organophosphorous insecti-cide, that was first registered as a broad spectrum insecticide in1965. It was used widely to control a variety of pests in agricultureand animal farm [5]. CPF interferes with the acetyl cholinesteraseenzyme, which is necessary for normal nerve transmission [6]. It

ll rights reserved.

transaminase; ALT, alaninedehydrogenase.

aculty of Science, Suez Canal

.S. El-Shenawy).

has been reported that it is linked to male and female genital defor-mities [7,8]. Additionally, the exposure of laboratory animals toCPF elicits a number of effects including hepatic [9,10] and testic-ular damage [1].

Propolis (CAS No. 9009-62-5) (sometimes also referred to as ‘beeglue’) is the generic name for the resinous substance collected byhoneybees from various plant sources. Propolis has been mainlyused as home remedies and a personal product since 300 BC [11].In China propolis has also been used as a Chinese traditional medi-cine for a long time. Extracts of propolis are receiving renewedattention worldwide because of their beneficial effects, amongthem, the effective antioxidant activity and a general ‘‘back to nat-ure trend’’ [12]. Propolis typically conserts of waxes, resins, water,inorganics, phenolics and essential oils [12], the exact compositionof which is dependent upon the source plant(s). Propolis containssome minerals such as Mg, Ca, I, K, Na, Cu, Zn, Mn and Fe as wellas some vitamins like B1, B2, B6, C and E, and a number of fatty acids.In addition, it contains some enzymes as succinic dehydrogenase,glucose-6-phosphatase, adenosine triphosphatase and acid phos-phatase [13]. Propolis also contains more than 300 biochemical con-stituents, including mostly a mixture of polyphenols, flavonoidaglycones, phenolic acid and their esters, and phenolic aldehydesand ketones, terpenes, sterols, vitamins, amino acids [14].

Propolis is a resinous natural product collected from cracks inthe bark of trees and leaf buds which are enriched with the salivaryenzymes of honeybees. It has gained popularity and was used

176 R.H. ElMazoudy et al. / Pesticide Biochemistry and Physiology 101 (2011) 175–181

extensively in healthy drinks and foods to improve well-being andprevent diseases such as inflammation, heart disease, diabetes andeven cancer. Propolis possesses several biological properties suchas anti-inflammatory, anticancer, antioxidant, antibiotic and anti-fungal activities [15,16].

Present study was therefore, undertaken to assess the effects ofCPF on testes, the main organ of male reproduction in rats. More-over, the present study was conducted to compare and evaluatethe protective effects of propolis in restoring the altered biochem-ical and histopathological alterations in the testes of male ratsexposed to CPF, which may have implications in managing humanswith accidental exposures to such compounds.

2. Materials and methods

2.1. Test insecticide

CPF technical grade (98%) was obtained from El-Helb Companyfor pesticides and chemicals, Egypt.

2.2. Drugs

Ethanolic extract of propolis was obtained from Sigma, St. Louis,MO, USA (P8904, EEP, pH 7.3).

2.3. Animals

Healthy adult male albino rats of the Wistar strain ( Rattusnorvegicus) with proven fertility, 4–5 months of age and weighing150–160 g, were supplied from the Animal Breeding House of theMedical Research Institute (MRI), Alexandria University, Alexan-dria, Egypt. Animals were maintained at the animal care facilityin the Zoology Department, Faculty of Science, in plastic cagesunder controlled temperature (23 ± 2 �C), 12-h light/dark cycleand 50 ± 5% relative humidity. Water and food were available adlabium. Rats were acclimatized to the laboratory environment fortwo weeks prior to the start of experiments.

2.4. Experimental design

After the period of acclimation, animals were divided into fourgroups with 25 animals in each. The first group was used as con-trol. The animals of control group were orally given corn oil(4 ml/kg). The second male group was orally treated with CPF(9 mg/kg b.w.); about 1/25 LD50. The third male group was orallytreated with propolis (50 mg/kg b.w.) and fourth group was treatedwith combination of CPF (9 mg/kg b.w.) and propolis (50 mg/kgb.w.). The selected dose of the CPF was based on studies of McColl-ister et al. [17]. The duration of the oral administration during theexperiments lasts for 70-day for completion of the spermatogeniccycle and maturation of sperms in epididymis [18].

2.5. Mating and fertility indexes

After the end of the treatment course, males of control andexperimental groups of treated rats (n = 25/group), were mated1:1 with untreated proven fertile, with regular estrus cycle,females for 5 days (complete one estrous cycle) [19]. Mating wasconfirmed by the presence of vaginal plugs or deposition of sper-matozoan at the vaginal orifice upon vaginal examination. Theday that a vaginal plug was found was considered day 0 of gesta-tion. Then mating and fertility indexes were estimated andrecorded.

2.6. Sperm quantity and quality

The left caudal epididymis was used for sperm motility andright caudal epididymis was used for sperm counts and morphol-ogy. Left caudal epididymis was first weighed, placed in a Petridish. An appropriate dilution (1:20) was made with physiologicalsaline (0.9% NaCl). Caudal epididymis was nicked in few sites witha scalpel blade and kept at 37 �C to release the spermatozoa fromthe tubules. The sperm suspension was examined within 5 minafter their isolation from epididymis. The counting of both motileand immotile sperms was done at 40� magnification. The calcu-lated results were finally expressed as percent motility [20]. Rightcaudal epididymis was weighed, diluted in 1:20 with physiologicalsaline (0.9% NaCl) solution in a petri dish and minced with a scalpelblade in the mid-to-distal region. Suspension was kept at 37 �C for5 min for the dispersion of sperm into medium. Sperm suspensionwas pipated very gently 20 times and placed in a hemocytometerand total number of the sperm head counted [20] at 40� magnifi-cation. Each sample was counted twice and means value was takenfor calculation.

2.7. Body and genital organ weight

Initial and final body weights of male rats were recorded andsubsequently weight changes were calculated. After fertility study,the rats were sacrificed by cervical dislocation. The testes andaccessory sex organs (seminal vesicles, prostates and epididymis)were dissected out, trimmed off the attached tissues and weighedindividually. Then, the organ/body weight ratio was calculated.Specimens of the testes were fixed immediately in Bouin’s fluidfor histological study.

2.8. Biochemical assays

At the end of the 70th day of the treatment course, blood sam-ples were collected from anaesthetized males of all groups bypuncturing the retro-orbital venous plexus with a fine sterilizedglass capillary tube into heparin-coated and dry tube. The gatheredblood left for 20 min at room temperature, then centrifuged at3000 rpm (600 g) for 10 min for the separation of sera. The serawere kept in a deep freezer (�20 �C) until analyses of certain bio-chemical parameters. The biochemical measurements were per-formed according to the details given in the kit’s instructions.

2.8.1. Enzymes activity determinationActivities of serum lactate dehydrogenase (LDH), alanine trans-

aminase (ALT), aspartate transaminase (AST) and alkaline phos-phatase (AlP), and the levels were measured spectrophotometeryusing commercial kits (Boehringer Mannheim, GmbH, Mannheim,Germany).

2.8.2. Testosterone determinationSerum samples of the treated male rats were used for estimat-

ing testosterone concentration using radioimmunoassay (RIA)method. After incubation, the liquid contents in the tubes werewithdrawn and the bound radioactivity was determined usinggamma counter according to method described by Shalaby et al.[21].

2.8.3. Determination of serum lipid profileSerum concentrations of total lipids (TL) were assayed by the

method of Knight et al. [22] and that of cholesterol and triglycer-ides (TG) were determined by the method Carr et al. [23]. Highdensity lipoprotein-cholesterol (HDL-c) and low density lipopro-tein-cholesterol (LDL-c) were determined according to the meth-ods of Warnick et al. [24] and Bergmenyer [25], respectively.

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Very low-density lipoprotein (VLDL) was calculated mathemati-cally by dividing the values of TG by a factor of 5 according toFriedewald et al. [26].

2.9. Histopathological examination

Testes of the treated rats were taken and fixed in Bouin’s solu-tion. The fixed specimens were then trimmed, washed and dehy-drated in ascending grades of alcohol. These specimens werecleared in xylene, embedded in paraffin, sectioned at 4–6 lmthickness and stained with Hematoxylen and Eosin (H&E) thenexamined microscopically according to Lillie [27].

2.10. Statistical analysis

Data are expressed as mean values ± SD (n = 10). Statisticalanalysis was performed using one-way analysis of variance(ANOVA) to assess significant differences among treatment groups.For each significant effect of treatment, the post hoc Tukey’s testwas used for comparisons. The criterion for statistical significancewas set at P < 0.05. All statistical analyses were performed usingSPSS statistical version 8 software package (SPSS� Inc., USA).

3. Results

3.1. Morbidity and mortality

Male rats orally administered to CPF (9 mg/kg b.w./day) for70 days have shown signs of toxicity such as salivation, diarrhea,nose and eye bleeding, tremor. No death was recorded throughoutthe experimental groups.

3.2. Body and organs weights

Body and organs weights of experimental male rats are pre-sented in Table 1. No significant change in body weight gain wasobserved in male rats treated with propolis as compared to con-trols. However, a significant decrease in the body weight gainwas observed in groups that treated with CPF alone or in combina-tion with propolis as compared to control or CPF rat, respectively.Absolute and relative reproductive organ weights (testes, epididy-mides, prostate, and seminal vesicles) were reduced dramaticallyin the groups treated with CPF or its combination with propolisas compared to the control or CPF group, respectively. However,no statistically significant differences in absolute and relative

Table 1Effect of oral administration of chlorpyrifos and/or propolis for 70 days on the body and r

Control CPF

Body weights (g)Initial 172.7 ± 1.97 174Final 219.5 ± 3.25 189Body weight change (%) 27.1 ± 0.48 8Organs weights (g)Testes 2.68 ± 0.04 1.8Epididymides 0.83 ± 0.01 0.6Prostate 0.56 ± 0.02 0.3Seminal vesicles 1.82 ± 0.08 0.9Relative organ weightsTestes 1.22 ± 0.02 0.9Epididymides 0.38 ± 0.01 0.3Prostate 0.26 ± 0.01 0.1Seminal vesicles 0.83 ± 0.012 0.5

The data are presented as means ± SE, n = 25.a Significant difference as compared with control group (P 6 0.05).b Significant difference as compared with chlorpyrifos group (P 6 0.05).

genital organs weights were noted in propolis treated group ascompared to the control rats.

3.3. Sperm characteristics

Data of sperm characteristics (Table 2) showed that treatmentof male rats with CPF significantly (P < 0.01) decreased spermcount and progressive motility (%), while increased immotile andmorphologically abnormal sperms as compared to control andpropolis groups (Table 2). Treatment with propolis alone showedno significant effects on sperm concentration and motility, whilecaused significant decrease in non-viability and abnormal spermcompared to control group. On the other hand, treatment withpropolis in combination with CPF caused significantly alleviatedthe decline in sperm concentration and motility, and significantlyincreased the percent of viability but decrease abnormality ofsperms compared to CPF group, and this means that propolis min-imized the reproductive toxicity of CPF.

3.4. Mating and fertility indices

Fertility indexes of the male rats given CPF at doses of 9 mg/kg/b.w. for 70 consecutive days were 54% compared to 100% in thecontrol normal group. Rats given propolis at 50 mg/kg/b.w. havefertility index of 100% while it was 70% in male rats given combi-nation of Propolis and CPF as shown in Table 3.

3.5. Biochemical changes

3.5.1. Serum ALT, AST, ALP and LDH activitiesThe results of enzymes activities of male rats are shown in

Table 4. Male rats exposed to CPF (9 mg/kg/day) showed anincrease in the serum enzyme activities of ALT, AST, ALP and LDHlevels comparable to control. In addition, propolis treated groupdid not show any changes as compared to control. Combinationtreatment (CPF with propolis) decreased the enzymes activity ascompared with rat that treated with CPF only.

3.5.2. Serum lipids profileThe lipid profile in serum of male rats was assayed showing

significant (P < 0.05) increases in total lipids, triglycerides, totalcholesterol, VLDL-C and low density lipoprotein cholesterol(LDL-c) in CPF treated group compared to the control group (Ta-ble 5), while the content of high density lipoprotein cholesterol(HDL-c) showed a significant (P < 0.05) decrease in CPF treated

elative sexual organs weight of male rats.

Propolis CPF + Propolis

.6 ± 2.96 176.8 ± 2.46 173.5 ± 2.90

.3 ± 2.72a 221.4 ± 3.90 201.7 ± 4.30b

.4 ± 0.216a 25.2 ± 0.43 16.3 ± 0.39b

5 ± 0.07 2.69 ± 0.16 2.31 ± 0.0426 ± 0.02 0.83 ± 0.01 0.75 ± 0.026 ± 0.02a 0.56 ± 0.01 0.47 ± 0.01b

8 ± 0.02a 1.86 ± 0.12 1.47 ± 0.01b

8 ± 0.02 1.21 ± 0.02 1.15 ± 0.025 ± 0.01 0.37 ± 0.02 0.37 ± 0.019 ± 0.01a 0.25 ± 0.01 0.23 ± 0.012 ± 0.01a 0.84 ± 0.01 0.73 ± 0.02b

Table 2Sperm characteristics in male rats after oral administration of chlorpyrifos and/or propolis for 70 days.

Sperm parameters Control CPF Propolis CPF + propolis

Count/epididymis (�106) 91.2 ± 1.97 56.1 ± 0.93a 90.4 ± 1.736 78.3 ± 1.39b

Motility (%)Progressive 59.4 ± 0.94 25.1 ± 0.41a 55.6 ± 0.93 42.8 ± 0.78b

Non-progressive 15.8 ± 0.42 29.3 ± 0.53a 17.1 ± 0.41 23.6 ± 0.53b

Immotile 24.7 ± 0.53 45.5 ± 0.79a 27.3 ± 0.58 33.5 ± 0.65b

Morphology (%)Normal 86.4 ± 1.59 62.4 ± 1.214a 85.3 ± 1.59 78.7 ± 1.64b

Abnormal head 5.2 ± 0.05 19.0 ± 0.21a 5.9 ± 0.12 10.2 ± 0.21b

Abnormal tail 7.6 ± 0.12 17.1 ± 0.24a 8.1 ± 0.41 9.9 ± 0.19b

Other abnormalities 0.7 ± 0.01 1.5 ± 0.02 0.7 ± 0.01 1.1 ± 0.12Total abnormalities 13.5 ± 0.47 37.6 ± 0.59a 14.7 ± 0.21 21.2 ± 0.48b

Viability (%) 88.5 ± 1.58 56.5 ± 1.24a 86.5 ± 1.39 73.1 ± 1.59b

The data are presented as means ± SE, n = 25.a Significant difference as compared with control group (P 6 0.05).b Significant difference as compared with chlorpyrifos group (P 6 0.05).

Table 3Functional fertility parameters of male rats after oral administration of chlorpyrifos and/or propolis for 70 days.

Control CPF Propolis CPF + Propolis

Number of males that used for mating 25 25 25 25Mating index (%) 25/25 (100) 13/25a (52) 25/25 (100) 20/25b (80)Fertility index (%) 25/25 (100) 7/13a (54) 25/25 (100) 14/20b (70)

Mating index (%) = Number of males inseminated females/total number of males cohabited with females � 100.Fertility index (%) = Number of cohabited females becoming pregnant/number of non pregnant with evidence of vaginal plug � 100.

a Significant difference as compared with control group (P 6 0.05).b Significant difference as compared with chlorpyrifos group (P 6 0.05).

Table 4Effect of chlorpyrifos alone and its combination with propolis on serum ALT, AST, ALP and LDH activities of male rats after 70 days of oral administration.

Enzymes activities (U/L) Control CPF Propolis CPF + propolis

ALT 88.51 ± 2.72 121.36 ± 4.50a 87.58 ± 2.20 99.14 ± 2.28b

AST 156.82 ± 5.14 188.41 ± 7.24a 155.97 ± 3.56 163.50 ± 3.95b

ALP 139.06 ± 6.21 185.92 ± 7.78a 138.92 ± 5.31 157.73 ± 5.44b

LDH 1259.25 ± 34.36 2394.36 ± 34.90a 1257.47 ± 31.20 1876.73 ± 33.79b

The data are presented as means ± SE, (n = 10).AST, aspartate transaminase; ALT, alanin transaminase; AlP, alkline phosphatase; LDH, lactate dehydrogenase.

a Significant difference as compared with control group (P 6 0.05).b Significant difference as compared with chlorpyrifos group (P 6 0.05).

Table 5Biochemical changes of serum lipids profile after oral administration of male rats with chlorpyrifos and/or propolis for 70 days.

Parameters (mg/dl) Control CPF Propolis CPF + propolis

TL 289.98 ± 7.15 336.01 ± 8.70a 288.23 ± 8.38 311.34 ± 9.87b

TG 124.60 ± 6.93 136.71 ± 5.31 125.13 ± 5.12 129.92 ± 4.49b

TC 102.67 ± 3.11 135.50 ± 3.22a 103.45 ± 3.11 119.47 ± 3.35b

HDL-c 43.31 ± 1.26 32.43 ± 0.94a 42.64 ± 1.25 39.86 ± 1.27b

LDL-c 34.44 ± 0.90 75.75 ± 2.52a 35.76 ± 1.02 54.83 ± 1.97b

VLDL 24.92 ± 0.76 27.34 ± 0.91a 25.03 ± 0.94 21.78 ± 0.83b

The data are presented as mean ± SE, (n = 10).TL, total lipid; TG, triglycerides; TC, total cholesterol; HDL-c, high density lipoprotein-cholesterol; LDL-c, low density lipoprotein-cholesterol; VLDL, volatile low densitylipoprotein.

a Significant difference as compared with control group (P 6 0.05).b Significant difference as compared with chlorpyrifos group (P 6 0.05).

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group compared to the control group (Table 5). On the otherhand, no significant difference (P > 0.05) was showed in the lipidprofile of propolis group compared to the control rats (Table 5).Treatment the rats with CPF and propolis showed a significant(P < 0.05) decrease in the lipid content compared to the CPF group(Table 5).

3.5.3. Serum testosteroneData presented in Fig 1 showed significant decrease in serum

testosterone concentration (P < 0.05) in rats treated with CPF com-pared to control. While, propolis significantly increased testoster-one and mitigated the negative effects for CPF treated group onthis parameter. Nevertheless, CPF administration alone lowered

Fig. 1. Serum testosterone of male rats after oral administered with chlorpyrifosand/or propolis for 70 days. The data are presented as means ± SD, (n = 10).aSignificant difference as compared with control group (P 6 0.05). bSignificantdifference as compared with chlorpyrifos group (P 6 0.05).

R.H. ElMazoudy et al. / Pesticide Biochemistry and Physiology 101 (2011) 175–181 179

the level of testosterone without propolis, but a combination of CPFand propolis recovered the testosterone levels.

3.6. Histopathology

Fig. 2A–D shows photomicrographs of testes from the differentexperimental groups. Histopathological examination of the testesof normal rats revealed active mature functioning seminiferoustubules associated with complete spermatogenital cell series(Fig. 2A). Moreover, some germ cells had small and darkly stainednuclei. Sections of testes of rats treated with propolis alone showed

Fig. 2. Photomicrograph of testicular histology of the (A) control rats showing nospermatocytes, spermatids are seen. (B) Testis of rats treated with 50 mg/kg b.w. propotreated with combination of CPF and propolis, showing mild changes in the cellular comshowing vacuolation and deterioration the germinal cells are abnormal and complete detubules were severely damaged. (L) Lumen, (G) germinal epithelium, (⁄) vacoulation, (I)

that they are less or more similar to the control sections (Fig. 2B).Testis of rats treated with CPF plus propolis revealed that itregained nearly its normal structure and remarkable restorationof the normal picture of seminiferous tubules was attained. Thegerm cells appeared regular in shape with disappearance of mostcytoplasmic vacuolation. Most nuclei became vesicular (Fig. 2C).However, the histological architecture of the testicular pathologyof CPF treated rats showed depression of spermatogenesis, Sertolicell toxicity and degeneration of somniferous tubules (Fig. 2D).The general architecture of some seminiferous tubules was disor-ganized. They were characterized by the accumulation of exfoli-ated germ cells within the affected tubules and appearance ofcytoplasmic vacuolation (Fig. 2D).

4. Discussion

The administration of chlorpyrifos brought about marked alter-ation in the weight of testes. The reduction in the testicular weightreflects regressive changes in seminiferous tubules. The change intesticular weight has also corresponded to the presence or absenceof post meiotic germ cell [1]. Reduction in the number of sper-matogenic elements and spermatozoa leads to reduction in theweight of testes. Similar reduction in the weight of testes was alsoobserved by Joshi et al. [1] with different doses and exposure timefrom the present study. Decrease in testicular weight was accom-panied by necrotic changes [28].

Sperm motility is an important functional measurement to pre-dict sperm fertilizing capacity. Any negative impact on motilitywould seriously affect fertilizing ability. In the present study,marked inhibition of sperm motility in CPF treated group may bebecause of low level of ATP content [29]. Sperm motility may beaffected by altered enzymatic activities of oxidative phosphorolyticprocess. Oxidative phosphorolytic process is required for ATP pro-duction, a source of energy for the forward movement of spermato-zoa [30]. Full ATP pool is crucial for normal spermatozoa movement

rmal structure of germinal epithelium with primary spermatocytes, secondarylis, showing the normal architecture of the seminiferous tubules. (C) Testis of rats

ponents of germinal epithelium. (D) Testis of rats treated with 9 mg/kg b.w. CPF,struction is seen in all components of the cells. The structures of the seminiferousintercellular spaces, original magnification 200�.

180 R.H. ElMazoudy et al. / Pesticide Biochemistry and Physiology 101 (2011) 175–181

and a slight deprivation of ATP leads to reduction in motility, whichmay cause infertility. Sperm count is considered to be one of theimportant factor that affect fertility [30]. Suppression of gonadotro-phins might have caused decrease in sperm density in testes [1].Also, toxicants have direct effect on sertoli cell function, whichappears to be involved in the control of spermiation, and when dis-turbed caused epithelial disorganization and subsequent tubularatrophy [31]. The negative fertility test may be attributed to lackof forward progression and reduction in density of spermatozoaand altered biochemical milieu of caudal epididymis. The presentstudy considers the first study that used propolis against the toxic-ity of CPF. The male rats that were given combination of propolisand CPF have fertility index 70% while the fertility index in the rattreated with CPF only was 54% as compared to control group.

In the present findings, increased level of cholesterol isattributed to decreased androgen concentration, which resultedin impaired spermatogenesis [31]. Similar effects by otherinsecticides have also been reported [1,3,32]. Reduction in theserum testosterone, clearly demonstrated the inhibitory effectof insecticides on the secretion of pituitary gonadotrophins(FSH and LH) and in turn on the testosterone biosynthesis inthe testes of rat [33]. Marked reduction in testosterone contentin association with highly reduced circulating levels of thishormone confirmed alteration in the reproductive physiology ofrat. The decrease in testosterone production may be inducedby the stimulation of P450 aromatase (P450arom), whichcatalyzes estrogen production from androgen, thereby decreasingandrogen levels [34]. These results suggest that chlorpyrifosexerts suppressive effects on testicular function and leads toinfertility in rats which in turn could be enhanced by usingpropolis.

The decrease in plasma testosterone levels, body weight, rela-tive testes and epididymis weights observed here confirms earlierresults of Grote et al. [35] in rats and Sarpa et al. [36] in mice. Sarpaet al. [36] found that treatment with triphenyltin chloride duringgestation days 6–17 of mice caused a decrease in weight gainand food intake. The decrease in relative testes and epididymisweights of rat treated with CPF agreed with results obtained byYousef et al. [37], who found that treatment with triphenyltin chlo-ride caused a decrease in relative weight of testis and epididymidesof rabbits. The present study declared that CPF caused a decrease inepididymal sperm count and sperm viability of rat. These resultscould be suggested that CPF impair male reproduction in rat bydecreasing circulatory testosterone. The decline in ejaculatevolume, sperm concentration, and total sperm output and semeninitial fructose concentration (Table 2) can be partly attributed tothe CPF-induced reduction in testosterone (Fig. 1). The observeddecrease in sperm motility could be attributed in part to the con-comitant abnormality of the sperms, decrease their viability (Table2) and the decrease in body weight (Table 1). Moreover, CPF hasbeen reported that it was able to generate reactive oxygen species(ROS) in different tissue organs [9,38] which coincides with theelevation in lipid peroxidation induced in seminal plasma (notpublished data). Overproduction of ROS can be detrimental tosperm as it is may be associated with male infertility [39]. Thus,the sperm toxic effect of triphenyltin chloride might be due toinduced free radicals.

The transaminases and phosphatase in semen play an impor-tant role in transamination and phosphorylation processes insperm metabolism [40]. The present results revealed a significant(P < 0.05) decrease in the activities of serum AST, ALT, LDH andAlP of rats treated with CPF. El-Kashoury and Tag El-Din [40]reported that CPF in different local manufactures (chlorozan,pestpan and pyriban) at dose of 23.43, 21.40 and 17.43 mg/kgb.w., respectively significant decrease the activities of ALP andLDH. This fact is a conventional indicator of liver injury due to

CPF-treatment [41]. Moreover, propetamphos (15 mg/kg b.w./day) was determined to cause harmful effects in rats by increas-ing the levels of glucose and TG, and the activities of AST, ALPand ALT after 28 of the treatment. Administration of propolis(100 mg/kg b.w./day) to these animals alleviated the harmful ef-fects of propetamphos [42].

The serum content of lipid profile showed remarkableincrease due to CPF-treatment (Table 5); significant increasesin TL, TG, TC and LDL-cholesterol were shown in CPF-treated ratscompared to the control group. Results of the present studyclearly indicate that previous administration of the Propolismarked change in lipoproteins and marked fall in the totalcholesterol and LDL-c in rats induced by CPF in the rat bloodserum.

In the current study we evaluated the protective role of propo-lis against the toxicity and the histological changes resulting fromthe administration of CPF in rats. Many environmental, physiolog-ical, and genetic factors have been implicated in defective spermfunction, the most common cause of infertility. Free radical-induced oxidative damage to spermatozoa is one such conditionwhich has recently gained a considerable attention for its role ininducing poor sperm function and infertility [43]. Factors thatcan offer spermatozoa protection are, therefore, of great impor-tance. In the present study, we found evidence suggesting thatpropolis possesses the capacity to protect sperm, testes andenzymes from deleterious actions of CPF. Propolis increased(P < 0.05) the levels of testosterone, relative testes and epididymisweight, and alleviated the negative effects of CPF, and this is inaccordance with Yousef et al. [40] who suggested that the increasein weight gain and feed intake of animals treated with propolisand triphenyltin due to high content of flavonoids. In vitro studyby Russo et al. [43] examined the effect of propolis on humanspermatozoa treated with benzo[a]-pyrene and exogenous reac-tive oxygen species (hydrogen peroxide; H2O2, adenosine50-diphosphate; ADP and ferrous sulfate; FeSO4). They demon-strated that propolis is able to protect genomic DNA by damage in-duced by benzo[a]-pyrene, H2O2 and H2O2 in combination withADP and FeSO4, determining a significant reduction of the intracel-lular oxidants. Bhadauria et al. (2007) reported that protectiveactivity of propolis at 200 mg/kg dose was statistically comparedwith positive control silymarin (50 mg/kg, po), a known hepato-protective drug seems to be better in preventing hepatic CYP2E1 activity deviated by CCl4. These results lead us to speculatethat propolis may play hepatoprotective role via improved CYP2E1 activity and reduced oxidative stress in living system [44].

The present study showed that the administration of propolisalone caused significant improvements in sperm characteristicsand male fertility of rats.

5. Conclusion

The present results showed that exposure to CPF caused deteri-oration in semen quality, decrease of fertility indexes as well asalternation in lipid profile and decreased the enzyme activities ofserum. Moreover, the testosterone decreased and there were manyhistological changes in the tests. The combination treatment ofpropolis with CPF had beneficial effects in improving the reproduc-tive performance of male rats and improved all the biochemicalparameters. Therefore, the present study elucidated the therapeu-tic effects of propolis administered in combination with CPF tominimize its reproductive toxicity.

Conflict of interest

The authors declare that there are no conflicts of interest.

R.H. ElMazoudy et al. / Pesticide Biochemistry and Physiology 101 (2011) 175–181 181

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